Abstract: Glycogen is a critical reserve of oxidizable fuel for brain neuron use during states of heightened activity or glucoprivation. Iatrogenic hypoglycemia is a recurring complication of obligatory meticulous control of insulin-dependent diabetes mellitus and poses a serious risk of neural injury. There is thus keen interest to maximize protection against harmful effects of hypoglycemia by cerebral glycogen. Our studies show that estradiol and hindbrain catecholamine neurotransmitter signals converge to regulate ventromedial hypothalamus nucleus (VMH) glycogen metabolic enzyme protein expression. This project will address the hypothesis that astrocyte adenosine 5?-mono-phosphate-activated protein kinase (AMPK) regulates VMH glycogen metabolism, and that estradiol acts via classical receptor-mediated signaling to control effects of basal and hypoglycemia-enhanced hindbrain noradrenergic input on sensor activity. Estrogen augmentation of the glycogen fuel reserve is presumed to correlate with prolonged local nerve cell energetic stability during hypoglycemia. Proposed research is guided by three Specific Aims: 1) Determine effects of estradiol on hypothalamic metabolic sensory structure glycogen content in normo- and hypoglycemic ovariectomized female rats; 2) Assess the role of VMH astrocyte estrogen receptor variants in AMPK- mediated glycogen metabolic responses to glucoprivation; and 3) Determine substrates for interaction of hindbrain catecholamine and estrogen regulation of VMH astrocyte glycogen metabolism. This project will use a combinatory in situ immunocytochemistry/laser-capture microdissection/high-sensitivity qPCR and Western blot approach for quantitative cell type-specific molecular profiling; pharmacologic tools for manipulation of glycogen metabolic enzyme activity; nano- technological devices for selective knockdown of astrocyte protein expression; paired glycogen densitometric histochemistry and nerve cell AMPK/phosphoAMPK Western blot analyses to correlate high-resolution quantitative glycogen maps with VMH metabolic sensory nerve cell AMPK activity; and scanning particle-induced X-ray emission spectrometry to perform single-cell microanalysis of electrolyte correlates of VMH neuron electrical activity, and to quantify effects of estradiol on hypoglycemic patterns of transynaptic anterograde transport of Mn, a reliable indicator of neuron activation, between hindbrain A2 noradrenergic neurons and the VMH. This work will utilize an innovative assembly of investigative strategies to identify cellular and molecular mechanisms of estrogen regulation of astrocyte glycogen mass, knowledge that can be leveraged to advance development of therapeutic strategies for neuro- protective amplification of brain glycogen levels.